Active Recombinant Human TFPI protein, His-tagged

• Cat.No.: TFPI-875H
• Product Overview: Active Recombinant Human TFPI protein(NP_006278.1) (Met 1-Lys 282) was expressed in HEK293 with a C-terminal polyhistidine tag.

Specification

• Species: Human
• Source: HEK293
• Tag: His
• Protein Length: 1-282 aa
• Form: Lyophilized from sterile PBS, pH 7.4.
• Bio-activity: Measured by its ability to inhibit trypsin cleavage of a fluorogenic peptide substrate, Mca-RPKPVE-Nval-WRK(Dnp)-NH2. The IC50 value is < 0.4 nM, as measured in 100μL reaction mixture containing 1.25 ng trypsin, 10 μM substrate, 50 mM Tris, 10 mM CaCl2, 0.15 M NaCl, 0.05% Brij-35, pH 7.5.
• Molecular Mass: The secreted recombinant human TFPI comprises 265 amino acids with a predicted molecular mass of 30.6 kDa. As a result of glycosylation, the apparent molecular mass of rhTFPI is approximately 42-45 kDa in SDS-PAGE under reducing conditions.
• Endotoxin: < 1.0 EU per μg protein as determined by the LAL method.
• Purity: > 85 % as determined by SDS-PAGE
• Storage: Store it under sterile conditions at -20°C to -80°C upon receiving. Recommend to aliquot the protein into smaller quantities for optimal storage. Avoid repeated freeze-thaw cycles.
• Reconstitution: It is recommended that sterile water be added to the vial to prepare a stock solution of 0.2 ug/ul. Centrifuge the vial at 4°C before opening to recover the entire contents.
• Publications:
  • MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia (2020)

Gene Information

• Gene Name: TFPI tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor) [ Homo sapiens ]
• Official Symbol: TFPI
• Synonyms: TFPI; tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor); LACI; tissue factor pathway inhibitor; EPI; extrinsic pathway inhibitor; TFI; TFPI1; anti-convertin
• Gene ID: 7035
• mRNA Refseq: NM_001032281
• Protein Refseq: NP_001027452
• MIM: 152310
• UniProt ID: P10646

Citation

MG1113, a specific anti–tissue factor pathway inhibitor antibody, rebalances the coagulation system and promotes hemostasis in hemophilia

Journal: Research and Practice in Thrombosis and Haemostasis    PubMed ID: 33313469    Data: 2020/10/22

Authors: Heechun Kwak, Sumin Lee, Sung Ho Hwang

Article Snippet:MG1113 and recombinant human (rh) TFPI (Creative BioMart, Shirley, NY, USA) were incubated at 37°C for 10 minutes. rhFXa (Enzyme Research Laboratories, South Bend, IN, USA) was then added and incubated at 37°C for 30 minutes, followed by the addition of FXa substrate, S‐2765 (Instrumentation Laboratory, Bedford, MA, USA).. The absorbance was measured using a VersaMax plate reader (Molecular Devices, San Jose, CA, USA) at 405 nm.The absorbance was measured using a VersaMax plate reader (Molecular Devices, San Jose, CA, USA) at 405 nm.

MG1113 binds to KD2 of tissue factor pathway inhibitor (TFPI). (A) TFPI expression vector was transfected into HEK 293 cells. Binding of Kunitz‐2 domain (KD2) of TFPI with MG1113 was then confirmed using immunoprecipitation (IP). A band was observed by western blot (WB) only in cases of TFPI constructs possessing KD2. Mock: human embryonic kidney (HEK) 293 cells were transfected without TFPI expression vector. (B) Two perpendicular views are shown with three polypeptides in different colors. Immunoglobulin (Ig) heavy chain is denoted by VH and CH. Ig kappa light chain is indicated by Vk and Ck. Mapping of epitopes and paratopes, defined as residues within the intersubunit distance of 4.5 ?, on surface representations of KD2 and Fab of MG1113. Left, epitopes on KD2 are shown in three different colors. Orange, yellow, and red indicate putative activated factor X (FXa)‐binding residues, which overlap with Fab of MG1113‐binding residues of KD2. Arg107 (in red) is a key residue in the inhibition of FXa. Right, paratopes on MG1113 are shown in magenta for VH residues and in pink for Vk residues. (C) MG1113 no longer binds to the construct from which the epitope of TFPI is removed. Ab, antibody; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase

Neutralizing effect of MG1113 on the function of tissue factor pathway inhibitor‐α (TFPI‐α). (A) The activity of 1 nM activated factor X (FXa) is reduced by 10 nM TFPI. The activity of FXa is recovered (n = 3) after treatment with MG1113 (0.625‐40 nM). (B) Activation of 10 nM factor X (FX) by extrinsic FXase, comprised of 10.5 pM tissue factor (TF) and 0.5 nM activated factor VII (FVIIa), is inhibited by 9 nM TFPI. Generation and activity of FXa are recovered (n = 4) after treatment with MG1113 (0.13‐800 nM). (C) After factor VIII (FVIII) deficient plasma is treated with MG1113 through thrombin generation assay, with increasing concentration, thrombin generation is also increased (n = 3). (D, E) In a modified prothrombin time (mPT) assay, when FVIII or factor IX (FIX) deficient plasma is treated with MG1113, with increasing concentration, clotting time is further shortened (n = 3). The graph represents mean and standard deviation

MG1113 not only binds to tissue factor pathway inhibitor‐β (TFPI‐β) but also neutralizes it. (A) Binding of human umbilical vein endothelial cells (HUVECs) and MG1113 is confirmed. Differently from human IgG (hIgG), which is a negative control, with increasing concentration of MG1113 (0.003 ‐ 200 nM), increased binding of MG1113 to HUVECs (n = 3) is observed. (B) The factor X (FX) is activated by activated factor VII (FVIIa) and tissue factor, which is expressed on stimulated HUVECs by tumor necrosis factor‐α. With increasing concentration of MG1113 (0.006‐500 nM), generation of activated factor X (FXa) is increased (n = 2). The graph represents mean and standard deviation